X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. The selective pressure for B cells expressing normal Btk suggests that introduction of a normal Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. Using onco-retroviral vectors optimized for murine HSC expression and transplantation of transduced stem cells into Btk/Tec doubly deficient recipients we have recently achieved: full rescue of both primary and peripheral Btk-dependent B-cell development, and correction of B cell functional responses. This data demonstrates that Btk gene transfer can reconstitute Btk-dependent functions in an animal model of XLA, and strongly support the further pursuit of this therapeutic approach. The current proposal seeks to address several key issues required to move forward with development of a definitive XLA genetic therapy including development of lentiviral vectors that: mediate highly efficient Btk gene delivery into human HSC; specifically target Btk expression to B lineage cells; and exhibit a significantly reduced risk of viral enhancer mediated mutagenesis proximal to the sited of viral integration. We will test the hypotheses that: 1) Lentiviral vectors containing B-lineage specific promoter/enhancers will mediate sustained, temporally appropriate levels Btk gene expression and lead to rescue of B cell function in vivo and in vitro; and that 2) Incorporation of an insulator elements into these vectors will promote both improved expression and, more importantly, a significantly reduced risk for of viral enhancer mutagenesis. Recent clinical trials for primary immunodeficiency disorders highlight the great potential for genetic correction as well as an unanticipated, high risk for development of onco-retroviral associated malignancies. While conceptually simple, implementation of definitive genetic therapy in XLA will require a concerted program of basic and applied research aimed at developing safe and efficient vector systems capable of appropriate, specific, and sustained B lineage gene expression. Overcoming these technical challenges should also provide insight for development of gene therapy in congenital diseases that lack a selective advantage.